This invention is in the area of methods for the treatment of hepatitis B virus (also referred to as xe2x80x9cHBVxe2x80x9d) that includes administering to a host in need thereof, an effective combination of nucleosides which have known anti-hepatitis B activity.
HBV is second only to tobacco as a cause of human cancer. The mechanism by which HBV induces cancer is unknown, although it is postulated that it may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis, and cell regeneration associated with the infection.
Hepatitis B virus has reached epidemic levels worldwide. After a two to three month incubation period in which the host is unaware of the infection, HBV infection can lead to acute hepatitis and liver damage, that causes abdominal pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive sections of the liver are destroyed.
Patients typically recover from acute hepatitis. In some patients, however, high levels of viral antigen persist in the blood for an extended, or indefinite, period, causing a chronic infection. Chronic infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. By mid-1991, there were approximately 225 million chronic carriers of HBV in Asia alone, and worldwide, almost 300 million carriers. Chronic persistent hepatitis can cause fatigue, cirrhosis of the liver, and hepatocellular carcinoma, a primary liver cancer.
In western industrialized countries, high risk groups for HBV infection include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is very similar to that of acquired immune deficiency syndrome (AIDS), which accounts for why HBV infection is common among patients with AIDS or AIDS related complex. However, HBV is more contagious than HIV.
However, more recently, vaccines have also been produced through genetic engineering and are currently used widely. Unfortunately, vaccines cannot help those already infected with HBV. Daily treatments with xcex1-interferon, a genetically engineered protein, has also shown promise, but this therapy is only successful in about one third of treated patients. Further, interferon cannot be given orally.
A number of synthetic nucleosides have been identified which exhibit activity against HBV. The (xe2x88x92)-enantiomer of BCH-189, known as 3TC, claimed in U.S. Pat. No. 5,539,116 to Liotta, et al., has been approved by the U.S. Food and Drug Administration for the treatment of hepatitis B. See also EPA 0 494 119 A1 filed by BioChem Pharma, Inc.
xcex22-Hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (xe2x80x9cFTCxe2x80x9d), claimed in U.S. Pat. Nos. 5,814,639 and 5,914,331 to Liotta, et al., exhibits activity against HBV. See Furman, et al., xe2x80x9cThe Anti-Hepatitis B Virus Activities, Cytotoxicities, and Anabolic Profiles of the (xe2x88x92) and (+) Enantiomers of cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]Cytosinexe2x80x9d Antimicrobial Agents and Chemotherapy, December 1992, page 2686-2692; and Cheng, et al., Journal of Biological Chemistry, Volume 267(20), 13938-13942 (1992).
U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu, et al.) disclose the use of 2xe2x80x2-fluoro-5-methyl-xcex2-L-arabinofuranolyluridine (L-FMAU) for the treatment of hepatitis B and Epstein Barr virus.
U.S. Pat. No. 5,767,122 to Emory University and The University of Georgia Research Foundation, Inc. discloses and claims enantiomerically pure xcex2-D-dioxolanyl nucleosides of the formula: 
wherein R is NH2, OH, Cl, or H. A method for treating HBV infection using a combination of DAPD and FTC is claimed in U.S. Pat. No. 5,684,010 to Raymond F. Schinazi.
Penciclovir (2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6H-purin-6-one; PCV) has established activity against hepatitis B. See U.S. Pat. Nos. 5,075,445 and 5,684,153.
Adefovir (9-[2-(phosphonomethoxy)ethyl]adenine, also referred to as PMEA or [[2(6-amino-9H-purin-9-yl)ethoxy]methylphosphonic acid), also has established activity against hepatitis B. See for example U.S. Pat. Nos. 5,641,763 and 5,142,051.
Yale University and The University of Georgia Research Foundation, Inc. disclose the use of L-FDDC (5-fluoro-3xe2x80x2-thia-2xe2x80x2,3xe2x80x2-dideoxycytidine) for the treatment of hepatitis B virus in WO 92/18517.
von Janta-Lipinski et al. disclose the use of the L-enantiomers of 3xe2x80x2-fluoro-modified xcex2-2xe2x80x2-deoxyribonucleoside 5xe2x80x2-triphosphates for the inhibition of hepatitis B polymerases (J. Med. Chem., 1998, 41,2040-2046). Specifically, the 5xe2x80x2-triphosphates of 3xe2x80x2-deoxy-3xe2x80x2-fluoro-xcex2-L-thymidine (xcex2-L-FTTP), 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-fluoro-xcex2-L-cytidine (xcex2-L-FdCTP), and 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-fluoro-xcex2-L-5-methylcytidine (xcex2-L-FMethCTP) were disclosed as effective inhibitors of HBV DNA polymerases.
It has been recognized that drug-resistant variants of HBV can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in the viral lifecycle, and most typically in the case of HBV, DNA polymerase. Recently, it has been demonstrated that the efficacy of a drug against HBV infection can be augmented by administering the compound in combination with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution, or other parameter of the drug can be altered by such combination therapy. In general, combination therapy induces multiple simultaneous stresses on the virus.
United U.S. Pat. No. 5,808,040 discloses that L-FMAU can be administered in combination with FTC, 3TC, carbovir, acyclovir, interferon, AZT, DDI (2xe2x80x2,3xe2x80x2-dideoxyinosine), DDC (2xe2x80x2,3xe2x80x2-dideoxycytidine), L-DDC, L-F-DDC, and D4T.
United U.S. Pat. No. 5,674,849 discloses the use of a nucleoside in combination with an oligonucleotide for the treatment of a viral disease.
U.S. Pat. No. 5,684,010 discloses a method for the treatment of hepatitis B that includes administering in combination or alternation a compound of the formula: 
wherein R is NH2, OH, or Cl, with FTC, 3TC, carbovir, or interferon.
WO 98/23285 discloses a method for the treatment or prophylaxis of hepatitis B virus infections in a human or animal patient which comprises administering to the patient effective or prophylactic amounts of penciclovir (or a bioprecursor thereof such as famciclovir) and alpha-interferon.
In light of the fact that hepatitis B virus has reached epidemic levels worldwide, and has severe and often tragic effects on the infected patient, there remains a strong need to provide new effective treatments for humans infected with the virus that have low toxicity to the host.
Therefore, it is an object of the present invention to provide new methods for the treatment of human patients or other hosts infected with hepatitis B virus and related conditions comprising administering a synergistically effective amount of a combination of anti-HBV agents.
It has been discovered that certain combinations of agents with hepatitis B activity are synergistic, and thus can provide enhanced benefits to the patient when administered in an effective combination or alternation dosage pattern.
In one preferred embodiment of the present invention, a method for treating HBV infection and related conditions in humans is disclosed, comprising administering a synergistically effective amount of xcex2-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), preferably substantially in the form of the (xe2x88x92)-optical isomer, or a pharmaceutically acceptable salt, ester or prodrug thereof with Penciclovir (2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6H-purin-6-one, also referred to as xe2x80x9cPCVxe2x80x9d). Famciclovir, or any other bioprecursor of Penciclovir, can be used in place of Penciclovir in any embodiment of this invention.
Another preferred embodiment of the present invention is a method for treating HBV infection and related conditions in humans, comprising administering in combination or alternation a synergistically effective amount of xcex2-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), preferably substantially in the form of the (xe2x88x92)-optical isomer, or a pharmaceutically acceptable salt, ester or prodrug thereof, with 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, also referred to below as Bis-POM-PMEA or BP-PMEA), or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier.
In another preferred embodiment of the present invention, a method for treating HBV infection and related conditions in humans is disclosed, comprising administering in combination or alternation a synergistically effective amount of 2xe2x80x2-fluoro-5-methyl-xcex2-L-arabinofuranolyluridine (L-FMAU), or a pharmaceutically acceptable salt, ester or prodrug thereof, with a compound of the formula: 
preferably xcex2-D-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine (DAPD), which is preferably administered in substantially pure form, or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier.
In yet another preferred embodiment of the present invention, a method for treating HBV infection and related conditions in humans is disclosed, comprising administering a synergistically effective combination or alternation amount of 2xe2x80x2-fluoro-5-methyl-xcex2-L-arabinofuranolyluridine (L-FMAU), or a pharmaceutically acceptable salt, ester or prodrug thereof, with Penciclovir, or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier.
In still another preferred embodiment of the present invention, a method for treating HBV infection and related conditions in humans is disclosed, comprising administering a synergistically effective amount of 2xe2x80x2-fluoro-5-methyl-xcex2-L-arabinofuranolyluridine (L-FMAU), or a pharmaceutically acceptable salt, ester or prodrug thereof, with 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier.
Another preferred embodiment of the present invention comprises a method for treating HBV infection and related conditions in humans, comprising administering a synergistically effective amount of a compound of the formula: 
wherein R is NH2, OH, H, or Cl (collectively referred to herein as the DAPD compounds), preferably, xcex2-D-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine (DAPD), which is preferably administered in substantially pure form, or a pharmaceutically acceptable salt, ester or prodrug thereof, with PMEA, or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier.
As used herein , the term xe2x80x9cisolated enantiomerxe2x80x9d refers to a nucleoside composition that includes approximately 95% to 100%, or more preferably, over 97% of a single enantiomer of that nucleoside.
The terms xe2x80x9csubstantially pure formxe2x80x9d or substantially free of its opposite enantiomer refers to a nucleoside composition of one enantiomer that includes no more than about 5% of the other enantiomer, more preferably no more than about 2%, and most preferably less than about 1% is present.
The synergistic combination of compounds or their pharmaceutically acceptable esters or salts, are also useful in the prevention and treatment of HBV infections and other related conditions such as anti-HBV antibody positive and HBV-positive conditions, chronic liver inflammation caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis, and fatigue. These synergistic formulations can also be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HBV antibody or HBV antigen positive or who have been exposed to HBV.
The active compound can be converted into a pharmaceutically acceptable ester by reaction with an appropriate esterifying agents, for example, an acid halide or anhydride. The compound or its pharmaceutically acceptable derivative can be converted into a pharmaceutically acceptable salt thereof in a conventional manner, for example, by treatment with an appropriate base. The ester or salt of the compound can be converted into the parent compound, for example, by hydrolysis.
The term xe2x80x9csynergistic combinationxe2x80x9d refers to a combination of drugs which produces a synergistic effect in vivo, or alternatively in vitro as measured according to the methods described herein.
The active compounds disclosed herein are therapeutic nucleosides or cyclic or acyclic nucleoside analogs with known activity against hepatitis B. It has been discovered that certain combinations of nucleosides provide an advantage over monotherapy, or over other combinations. Not all combinations of the known anti-HBV drugs provide a benefit; it is often the case that drugs act antagonistically.
The active compound can be administered as any derivative that upon administration to the recipient, is capable of providing directly or indirectly, the parent compound, or that exhibits activity itself. Nonlimiting examples are the pharmaceutically acceptable salts (alternatively referred to as xe2x80x9cphysiologically acceptable saltsxe2x80x9d), and the 5xe2x80x2 and N4 cytosinyl or N6-adeninyl acylated (esterified) derivatives of the active compound (alternatively referred to as xe2x80x9cphysiologically active derivativesxe2x80x9d). In one embodiment, the acyl group is a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted with halogen, C1 to C4 alkyl or C1 to C4 alkoxy, or is a sulfonate ester such as alkyl or aralkyl sulphonyl including methanesulfonyl, phosphate, including but not limited to mono, di or triphosphate ester, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g., dimethyl-5-butylsilyl) or diphenylmethylsilyl. Aryl groups in the esters optionally comprise a phenyl group.
Modifications of the active compound, and especially at the N4 cytosinyl or N6 adeninyl and 5xe2x80x2-O positions, can affect the bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species. Further, the modifications can affect that antiviral activity of the compound, in some cases increasing the activity over the parent compound. This can easily be assessed by preparing the derivative and testing its antiviral activity according to the methods described herein, or other methods known to those skilled in the art.
Prodrugs
Any of the anti-hepatitis B agents described herein can be administered as a prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the nucleoside. A number of hydroxyl-bound prodrug ligands are known. In general, alkylation, acylation or other lipophilic modification of the hydroxy, mono, di or triphosphate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that can replace one or more hydrogens on the hydroxyl or phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect.
Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside, preferably at the 5xe2x80x2-OH of the nucleoside or hydroxyl of the acyclic nucleoside analogs (such as PMEA or Penciclovir), include U.S. Pat. Nos. 5,149,794 (Sep. 22, 1992, Yatvin, et al.); U.S. Pat. No. 5,194,654 (Mar. 16, 1993, Hostetler, et al.); U.S. Pat. No. 5,223,263 (Jun. 29, 1993, Hostetler, et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvin, et al.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler, et al.); U.S. Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler, et al.); U.S. Pat. No. 5,543,389 (Aug. 6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,390 (Aug. 6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvin, et al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996, Basava, et al.), all of which are incorporated herein by reference.
Foreign patent applications that disclose lipophilic substituents that can be attached to the active compounds of the present invention, or lipophilic preparations, include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.
The therapeutic nucleosides used in the synergistic compositions of the present invention and processes for preparing them are known in the art.
xcex2-2-Hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), and its enantiomers, can be prepared by the methods disclosed in U.S. Pat. Nos. 5,204,466, 5,700,937, 5,728,575 and 5,827,727, all of which are incorporated by reference.
2xe2x80x2-Fluoro-5-methyl-xcex2-L-arabinofuranolyluridine (L-FMAU) can be prepared by the methods disclosed in U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362 to Chu, et al. All of these patents are incorporated by reference.
Methods for the preparation of the DAPD compounds, including (2R,4R)-2-amino-9-[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine (DAPD) are disclosed in U.S. Pat. Nos. 5,767,122; 5,684,010; 5,444,063, and 5,179,104, all of which are incorporated by reference.
Pencyclovir can be prepared by the methods disclosed in U.S. Pat. Nos. 5,075,445 and 5,684,153.
PMEA can be prepared by the methods disclosed in U.S. Pat. Nos. 5,641,763 and 5,142,051.
Mono, di, and triphosphate derivatives of the active nucleosides can be prepared as described according to published methods. The monophosphate can be prepared according to the procedure of Imai, et al., J. Org. Chem., 34(6), 1547-1550 (June 1969). The diphosphate can be prepared according to the procedure of Davisson, et al., J. Org. Chem., 52(9), 1794-1801 (1987). The triphosphate can be prepared according to the procedure of Hoard, et al., J. Am. Chem. Soc., 87(8), 1785-1788 (1965).
It has been recognized that drug-resistant variants of HBV can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in the viral lifecycle, and most typically in the case of HBV, DNA polymerase. Recently, it has been demonstrated that the efficacy of a drug against HBV infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution, or other parameter of the drug can be altered by such combination therapy. In general, combination therapy induces multiple simultaneous stresses on the virus.